Synthesis of a tetrasaccharide fragment of mycobacterial arabinogalactan

The arabinogalactan of mycobacteria contains both monosaccharides in the furanose ring form, which are absent in mammals. We report here the first synthesis of the tetrasaccharide fragment alpha-D-Araf-(1-->5)-beta-D-Galf-(1-->5)-beta-D-Galf-(1-->6)-D-Galf, conveniently derivatized for further elongation. The strategy relied on the use of suitably substituted D-galactono-1,4-lactones as precursors for the galactofuranose units. Reduction of lactone tetrasaccharide 9 with disiamylborane afforded the tetrasaccharide synthon 1. The tetrasaccharide contains the linker unit of the arabinan to the galactan.     

Antitumour activity of purified arabinogalactan-peptidoglycan complex of the cell wall skeleton of Rhodococcus lentifragmentus

Antitumour activity of arabinogalactan peptidoglycan (AP) complex (peptidoglycan and arabinogalactan liberated by an acid or alkaline treatment from Rhodococcus lentifragmentus AN-115 cell wall skeleton) was examined in mice and compared with that of the cell wall skeleton. The growth of syngeneic fibrosarcoma Meth A cells after implantation in BALB/c mice was significantly suppressed by AP complex, and also regressed after intratumoral injection of AP complex on days 1, 4 and 7 after tumour implantation. Although the activity of peptidoglycan was less than that of AP complex, peptidoglycan also showed both tumour-suppressive and regressive activities. Arabinogalactan did not show antitumour activity. It is interesting that peptidoglycan has an important role in the effect against tumours.     

Analogues of the mycobacterial arabinogalactan linkage disaccharide as cell wall biosynthesis inhibitors

The mycobacterial arabinogalactan linkage disaccharide [alpha-L-Rha-(1-->3)-alpha-D-GlcNAc] provides a basis for the design of new antitubercular drugs, since it supports a key skeletal structure in the bacterial cell wall. A series of analogues of the linker was synthesized by coupling appropriate thiorhamnosyl donors modified at their 4-positions, with an N-acetyl glucosamine acceptor. In a cell-free enzyme inhibition assay, three analogues inhibited the activity of the galactosyltransferase that adds a Galf residue to the linkage disaccharide. Although the compounds were modest inhibitors, these data confirm the viability of this approach to anti-mycobacterial agents. It is especially significant that the three effective compounds are modified at the site of the acceptor atom in the natural substrate.     

Biosynthesis of the arabinogalactan-peptidoglycan complex of Mycobacterium tuberculosis

The compositional complexity of the mycobacterial cell envelope differentiates Mycobacterium species from most other prokaryotes. Historically, research in this area has focused on the elucidation of the structure of the mycobacterial cell envelope with the result that the structures of the mycolic acid-arabinogalactan-peptidoglycan complex from M. tuberculosis are fairly well understood. However, the current impetus for studying M. tuberculosis and other pathogenic mycobacteria is the need to identify targets for the development of new drugs. Therefore, emphasis has been shifting to the study of cell envelope biosynthesis and the identification of enzymes that are essential to the viability of M. tuberculosis. The publication of the complete M. tuberculosis genome in 1998 has greatly aided these studies. To date, thirteen enzymes involved in the synthesis of the arabinogalactan-peptidoglycan complex of M. tuberculosis have been identified and at least partially characterized. Eleven of these enzymes were reported subsequent to the publication of the M. tuberculosis genome, a clear indication of the rapid evolution of knowledge stimulated by the sequencing of the genome. In this article we review the current understanding of M. tuberculosis arabinogalactan-peptidoglycan structure and biosynthesis.     

The phosphate diester linkage of the peptidoglycan polysaccharide moieties of Micrococcus lysodeikticus cell wall

The external polysaccharide is a major component of Micrococcus lysodeikticus cell wall and displays distinct composition. The complete structure of the external polysaccharide had been elucidated as a basis for investigation of the cell wall structure-function relation. However, the mode of attachment of the polysaccharide to the peptidoglycan through a phosphodiester was not clear due to limitations in structural and biosynthetic studies. The present study describes purification of a lysozyme-resistant nondialyzable high-molecular-weight fragment of cell wall and identifies the sugar, D-glucose, as the point of external polysaccharide attachment to the peptidoglycan through a phosphate diester. Kinetic studies for the acid-catalyzed release of external polysaccharide from the peptidoglycan were performed in parallel with synthetic [methyl-2-acetamido-3-O-(D-1-carboxyethyl)-2-deoxy-alpha-D- glucopyranoside-6-yl]-alpha-D-glucopyranosyl phosphate and alpha-D-glucopyranosyl phosphate and showed the presence of a phosphodiester linkage between external polysaccharide and peptidoglycan. In addition, type of phosphate residue and cross-linking between muramic acid and protein part have been determined.     

Galactosyl transferases in mycobacterial cell wall synthesis

Two galactosyl transferases can apparently account for the full biosynthesis of the cell wall galactan of mycobacteria. Evidence is presented based on enzymatic incubations with purified natural and synthetic galactofuranose (Galf) acceptors that the recombinant galactofuranosyl transferase, GlfT1, from Mycobacterium smegmatis, the Mycobacterium tuberculosis Rv3782 ortholog known to be involved in the initial steps of galactan formation, harbors dual β-(1→4) and β-(1→5) Galf transferase activities and that the product of the enzyme, decaprenyl-P-P-GlcNAc-Rha-Galf-Galf, serves as a direct substrate for full polymerization catalyzed by another bifunctional Galf transferase, GlfT2, the Rv3808c enzyme.     

A transition state analog of lysozyme catalysis prepared from the bacterial cell wall tetrasaccharide.

Treatment of the cell wall tetrasaccharide GlcNAcbeta(1 leads to 4)-MurNAc-beta(1 leads to 4)-GlcNAc-beta(1 leads to 4)-MurNAc with alkali resulted in the formation of the unsaturated tetrasaccharide GlcNAc-beta(1 leads to 4)-MurNAc-beta(1 leads to 4)-GlcNAc-beta(1 leads to 4)-delta2,3-2-acetamido-2-deoxy-D-glucoseen. The same compound was also formed by transglycosylation upon incubation of the unmodified tetrasaccharide with the unsaturated disaccharide GlcNAc-beta(1 leads to 4)-delta2,3-2-acetamido-2-deoxy-D-glucoseen (Tipper, D. J. (1968) Biochemistry 7, 1441-1449) and hen egg white lysozyme. The unsaturated tetrasaccharide was further characterized by paper electrophoresis, amino sugar analysis, and NMR. From NMR analysis it is concluded that the delta2,3-2-acetamido-2-deoxy-D-glucoseen at the reducing end of the unsaturated tetrasaccharide has a half-chair conformation. This conformation is similar to the one proposed for the sugar at subsite D in the lysozyme-substrate complex in the transition state. Addition of the unsaturated tetrasaccharide to a solution of hen egg white lysozyme quenched the fluorescence of the enzyme and shifted the fluorescence maximum to the blue, similar to the effect produced by the parent compound. The association constant of the unsaturated tetrasaccharide and lysozyme was measured at pH 6.0 and 24 degrees by spectrofluorimetry and microcalorimetry and found to be 1.45 X 10(5) M-1 and 2.5 X 10(5) M-1, respectively. The average value is 100 times higher than that found for the binding of unmodified tetrasaccharide to the enzyme under the same conditions. The unsaturated tetrasaccharide proved to be a better inhibitor of the lysis of Micrococcus luteus cells than the parent compound by a factor of 35. These results support the hypothesis that the active site of the enzyme is constructed so as to bind the transition state for the reaction it catalyzes more firmly than the substrate itself.     

Modulation of interleukin-12 synthesis by DNA lacking the CpG motif and present in a mycobacterial cell wall complex

 A mycobacterial cell wall complex prepared from the non-pathogenic microorganism Mycobacterium phlei, where mycobacterial DNA is preserved and complexed to cell wall fragments, possesses anticancer and immunomodulatory activity. DNA from a number of prokaryotes has been found to modulate the immune system and to induce cytokine synthesis. We have therefore determined whether the DNA associated with this complex has the ability to induce the synthesis of interleukin-12 (IL-12), a potent anticancer cytokine. Mycobacterial DNA complexed with cell wall fragments or DNA purified from M. phlei induced IL-12 synthesis by murine and human monocytes and macrophages in vitro, and was capable of inducing IL-12 synthesis in vivo in mice following i.p. administration. Neutralization of DNA with cationic liposomes or digestion with DNase I significantly decreased the ability of the cell wall complex to induce IL-12. CpG methylation of DNA extracted from these cell walls or from M. phlei did not affect the induction of IL-12 synthesis by monocytes and macrophages. In contrast, CpG methylation of DNA from Escherichia coli abolished its ability to induce IL-12 synthesis. These results demonstrate that unmethylated CpG motifs present in M. phlei DNA are not a prerequisite for the induction of IL-12 synthesis. The size of the mycobacterial DNA, in the range of 5 bp to genomic DNA, did not influence its capacity to induce IL-12. Our results emphasize that M. phlei DNA associated with the cell wall complex makes a significant contribution to the overall immunomodulatory and anticancer activity of this mycobacterial cell wall preparation and that these activities are not correlated with the presence of CpG motifs. Received: 23 November 1999 / Accepted: 28 March 2000     

Synthesis and TNF-alpha inducing activities of mycoloyl-arabinan motif of mycobacterial cell wall components

The extract of the cell wall skeleton of Bacillus Calmette-Guérin (BCG-CWS) from Mycobacterium bovis is known to be an activator of innate immunity. Synthesis of pentaarabinofuranoside as part of the arabinan moiety of BCG-CWS was achieved by double alpha-arabinofuranosylation followed by double beta-arabinofuranosylation with orthogonally protected donors. Mycolic esters of the arabinan in the terminal lipo-arabinan motif of BCG-CWS were synthesized through alkylation of unprotected mycolic acid with bis- and tetra-tosylates of pentaarabinofuranoside. A series of compounds were subjected to a tumor necrosis factor alpha (TNF-alpha) secretion-inducing assay, disclosing aspects of the structure-activity relationship which should be useful in finding the site of the activity.     

Synthesis of Arabino glycosyl triazoles as potential inhibitors of mycobacterial cell wall biosynthesis

A series of arabino glycosyl triazoles with varying hydrophobic groups were synthesised as putative mimics of decaprenolphosphoarabinose (DPA) as potential inhibitors of mycobacterial cell wall biosynthesis. Biological testing against Mycobacterium bovis BCG revealed low to moderate anti-mycobacterial activity, with strong dependence on the identity of the hydrophobic side chain.     

Biosynthesis of the galactan component of the mycobacterial cell wall

The structural core of the cell walls of Mycobacterium spp. consists of peptidoglycan bound by a linker unit (-alpha-L-Rhap-(1-->3)-D-GlcNAc-P-) to a galactofuran, which in turn is attached to arabinofuran and mycolic acids. The sequence of reactions leading to the biogenesis of this complex starts with the formation of the linker unit on a polyprenyl-P to produce polyprenyl-P-P-GlcNAc-Rha (Mikusová, K., Mikus, M., Besra, G. S., Hancock, I., and Brennan, P. J. (1996) J. Biol. Chem. 271, 7820-7828). We now establish that formation of the galactofuran takes place on this intermediate with UDP-Galf as the Galf donor presented in the form of UDP-Galp and UDP-Galp mutase (the glf gene product) and is catalyzed by galactofuranosyl transferases, one of which, the Mycobacterium tuberculosis H37Rv3808c gene product, has been identified. Evidence is also presented for the growth of the arabinofuran on this polyprenyl-P-P-linker unit-galactan intermediate catalyzed by unidentified arabinosyl transferases, with decaprenyl-P-Araf or 5-P-ribosyl-PP as the Araf donor. The product of these steps, the lipid-linked-LU-galactan-arabinan has been partially characterized in terms of its heterogeneity, size, and composition. Biosynthesis of the major components of mycobacterial cell walls is proving to be extremely complex. However, partial definition of arabinogalactan synthesis, the site of action of several major anti-tuberculosis drugs, facilitates the present day thrust for new drugs to counteract multiple drug-resistant tuberculosis.     

First synthesis of the immunodominant beta-galactofuranose-containing tetrasaccharide present in the cell wall of Aspergillus fumigatus

β-Galf-(1→5)-β-Galf-(1→6)-α-Manp-(1→6)-α-Manp, the immunodominant epitope in the cell-wall galactomannan of Aspergillus fumigatus, was synthesized for the first time as its allyl glycoside. The key disaccharide glycosyl donor, 2,3,5,6-tetra-O-benzoyl-β-d-galactofuranosyl-(1→5)-2-O-acetyl-3,6-di-O-benzoyl-β-d-galactofuranosyl trichloroacetimidate (10), was constructed by 5-O-glycosylation of 1,2-O-isopropylidene-3,6-di-O-benzoyl-α-d-galactofuranose (4) with 2,3,5,6-tetra-O-benzoyl-β-d-galactofuranosyl trichloroacetimidate (5), followed by 1,2-O-deacetonation, acetylation, selective 1-O-deacetylation, and trichloroacetimidation. The target tetrasaccharide 16 was obtained by the condensation of allyl 2,3,4-tri-O-benzoyl-α-d-mannopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-α-d-mannopyranoside (14) as glycosyl acceptor with the disaccharide glycosyl donor 10, followed by deprotection.     

Identification of a novel galactosyl transferase involved in biosynthesis of the mycobacterial cell wall

The possibility of the Rv3782 protein of Mycobacterium tuberculosis being a putative galactosyl transferase (GalTr) implicated in galactan synthesis arose from its similarity to the known GalTr Rv3808c, its classification as a nucleotide sugar-requiring inverting glycosyltransferase (GT-2 family), and its location within the "possible arabinogalactan biosynthetic gene cluster" of M. tuberculosis. In order to study the function of the enzyme, active membrane and cell wall fractions from Mycobacterium smegmatis containing the overexpressed Rv3782 protein were incubated with endogenous decaprenyldiphosphoryl-N-acetylglucosaminyl-rhamnose (C(50)-P-P-GlcNAc-Rha) as the primary substrate for galactan synthesis and UDP-[(14)C]galactopyranose as the immediate precursor of UDP-[(14)C]galactofuranose, the ultimate source of all of the galactofuranose (Galf) units of galactan. Obvious increased and selective synthesis of C(50)-P-P-GlcNAc-Rha-Galf-Galf, the earliest product in the pathway leading to the fully polymerized galactan, was observed, suggesting that Rv3782 encodes a GalTr involved in the first stages of galactan synthesis. Time course experiments pointed to a possible bifunctional enzyme responsible for the initial synthesis of C(50)-P-P-GlcNAc-Rha-Galf, followed by immediate conversion to C(50)-P-P-GlcNAc-Rha-Galf-Galf. Thus, Rv3782 appears to be the initiator of galactan synthesis, while Rv3808c continues with the subsequent polymerization events.     

Synthesis of the tetrasaccharide core region of antigenic lipo-oligosaccharides characteristic of Mycobacterium kansasii

The oligosaccharide core region, beta-D-Glcp-(1----3)-beta-D-Glcp-(1----4)-alpha-D-Glcp- 1----1)-alpha-D-Glcp (1), of the lipo-oligosaccharide-type antigens isolated from M. kansasii has been synthesised from 2,3,2',3',4',6'-hexa-O-benzyl-6-O-(1-phenylethyl)-alpha, alpha-trehalose (4). Compound 4 was obtained by LiAlH4-AlCl3-type hydrogenolysis of 2,3,2',3',4',6'-hexa-O-benzyl-4,6-O-(S)-(1-phenylethylidene)-alpha , alpha-trehalose. The beta-laminaribiosyl part of the molecule was built-up by sequential glycosylation steps using 2,4,6-tri-O-acetyl-3-O-allyl-alpha-D-glucopyranosyl bromide in the presence of HgBr2 and methyl 2,3,4,6-tetra-O-acetyl-1-thio-beta-D-glucopyranoside promoted by methyl triflate. The complete a priori 13C-n.m.r. spectrum assignment of 1 was achieved by applying 2D methods.     

Synthesis and biological evaluation of trehalose analogs as potential inhibitors of mycobacterial cell wall biosynthesis

Analogs of trehalose are reported that were designed to interfere with mycolylation pathways in the mycobacterial cell wall. Several derivatives of 6,6'-dideoxytrehalose, including N,N'-dialkylamino and 6,6'-bis(sulfonamido) analogs, were prepared and evaluated for antimycobacterial activity against Mycobacterium tuberculosis H(37)Ra and a panel of clinical isolates of Mycobacterium avium. 6,6'-Diaminotrehalose and its diazido precursor were both inactive, but significant activity apparently related to aliphatic chain length was found among the sulfonamides, N-alkylamines, and one of the amidines.     

Synthesis of a tetrasaccharide substrate of heparanase

A tetrasaccharide, corresponding to the heparan sulfate heparanase substrate, namely β-d-GlcA(2S)-(1→4)-α-d-GlcN(NS,6S)-(1→4)-β-d-GlcA-(1→4)-α-d-GlcN(NS,6S)-OMe, was synthesized in a convergent manner via coupling of a pair of the disaccharide building blocks as a key step.